CRISPR-Cas9 has been explored as a transformative genome engineering tool for many eukaryotic organisms. However, its utilization in bacteria remains limited and ineffective. This chapter, taking Clostridium beijerinckii as an example, describes the use of Streptococcus pyogenes CRISPR-Cas9 system guided by the single chimeric guide RNA (gRNA) for diverse genome-editing purposes, including chromosomal gene deletion, integration, single nucleotide modification, as well as "clean" mutant selection. The general principle is to use CRISPR-Cas9 as an efficient selection tool for the edited mutant (whose CRISPR-Cas9 target site has been disrupted through a homologous recombination event and thus can survive selection) against? the wild type background cells. This protocol is broadly applicable to other microorganisms for genome-editing purposes.
Keywords: CRISPR-Cas9; Clostridium; Genome engineering; Homologous recombination; Single nucleotide modification (SNM); Synthetic biology.